Escherichia coli (E. coli) remains a safety concern in the preservation and quality of green leafy vegetables. Sugar–lectin interactions provide a reliable, specific, and effective sensing platform for the detection of bacteria as compared to the tedious conventional plate counting technique. Herein, we present the synthesis of 4-(N-mannosyl) benzoic acid (4-NMBA) and 4-thiophenyl-N-mannose (4-TNM) via a two-step reductive amination for the detection of E. coli using a quartz crystal microbalance (QCM) biosensor. The 4-NMBA was synthesized with mannose and para-aminobenzoic (4-PBA), while the 4-TNM was synthesized with mannose and 4-aminophenyl disulfide (4-AHP) using water and acetic acid in a 1:1 ratio. The resultant structure of mannose derivatives (4-NMBA and 4-TNM) was characterized and confirmed using analytical tools, such as Mass Spectrometer, SEM, and FTIR. The choice of ligands (mannose derivatives) is ascribed to the specific recognition of mannose to the FimH lectin of the type 1 pilus of E. coli. Furthermore, the 4-PBA and 4-AHP conjugated to mannose increase the ligand affinity to FimH lectins. The setup of the QCM biosensor was composed of modification of the crystal surface and the covalent attachment of ligands for the detection of E. coli. The piezoelectric effect (frequency shift of the quartz) was proportional to the change in mass added to the gold crystal surface. Both the 4-NMBA- and 4-TNM-coated QCM sensors had a limit of detection of 3.7 CFU/mL and 6.6 CFU/mL with a sensitivity of 2.56 × 103 ng/mL and 8.99 × 10−5 ng/mL, respectively, within the dynamic range of 103 to 106 CFU/mL. This study demonstrates the application of ligand-coated QCM biosensors as a cost-effective, simple, and label-free technology for monitoring pathogenic bacteria via molecular interactions on crystal surfaces. Pathogenic  E. coli is a commensal microorganism that naturally inhabits the intestinal tracts of warm-blooded animals to facilitate vitamin B12 synthesis [E. coli (STEC) is an infectious strain of E.coli that causes pediatric hemolytic uremic syndrome (HUS) and acute gastroenteritis by the consumption of contaminated food and water [Escherichia coli O104:H4, which recorded 810 cases and 39 deaths [E. coli can cause detrimental effects in humans [E. coli in contaminated food samples and water. The goal of this study is to design a mannose derivative for the detection of E. coli via a QCM biosensor. Pathogenic bacteria pose a threat to food quality and water systems globally. nd water . The casn humans ,6. TheseE. coli that binds to mannose [Bacteria toxins bind to the carbohydrate derivatives present on cell surfaces. The adherence and binding affinity of bacteria to human cells is the beginning of their pathogenesis . The att mannose ,9. Cumul mannose ,10. This mannose , ELISA [ mannose , and ele mannose  are a fe mannose . The limE. coli. The sensor was prepared with ssDNA probes complementary to a target DNA combined with nanoparticle amplification [3O4 nanoparticles via streptavidin to increase the frequency change with promising detection limits and sensitivity [Quartz crystal microbalance (QCM) is one method that has the potential for rapid and inexpensive detection . Howeverfication . The worsitivity ,20.E. coli via interaction with the FimH lectin, using QCM as a detection platform. First, 4-NMBA was synthesized using mannose and para-aminobenzoic acid, while the 4-TNM was synthesized using mannose and 4-aminophenyl disulfide [In this work, we demonstrated the synthesis of mannose-derived ligands (4-NMBA and 4-TNM have) for detecting isulfide . The ligisulfide ,22, we cEscherichia coli (25922) were purchased from ATCC . A quartz crystal analyzer model QCA917 from Seiko EG&G was used for all measurements. Furthermore, 10 MHz quartz crystals plated with gold were purchased from Princeton Applied Research  All reagents were analytical grade. Aqueous solutions were prepared with nano-pure water with 18.2 MΩ specific resistance.D (+)-mannose, 11-mercaptoundecanoic acid(MUA), 4-aminophenyl disulfide, and tryptic soy broth were purchased from Sigma . Para-Aminobenzoic acid and N-(3-dimethyl aminopropyl)-N′-ethyl carbodiimide (EDC) were purchased from Fisher Scientific . The borane–dimethylamine complex was purchased from Acros Organic . N-hydroxysuccimide (NHS) was purchased from Pierce Chemical Company . Mannose derivative (4-NMBA) was synthesized by reacting the aldehyde group of D (+)-mannose and the amino group of para-aminobenzoic acid (reductive amination). Briefly, 20 mM of D (+)-mannose was dissolved in a 1:1 ratio of ultrapure water and acetic acid. Once completely dissolved, 22 mM of p-aminobenzoic acid was added to the reaction. The solutions were mixed and incubated in a sealed flask for 1 h at 20 °C. Amination of the reactants was achieved following this step. After incubation, a 50 mM borane–dimethylamine reducing agent was added to the reaction and incubated overnight to achieve the reduction step. The resulting product was filtered and washed with pure water to remove unreacted reagents. The final product was crystallized. A similar procedure was adapted for synthesizing 4-TNM using 4-aminophenyl disulfide, for which 11 mM concentration was utilized. The names and identities of the synthesized ligands were predicted using ChemDraw and confirmed with mass spectrometry .Mass spectrometry characterization was performed for the mannose ligands to confirm the molecular structures. Both ligands were dissolved in liquid chromatography (LC) grade 50:50 mixture of acetic acid and water, followed by ESI-MS/MS analysis  ,23 to deE. coli, and inserted into a cassette connected to the quartz crystal analyzer. Both frequency shifts of the sensor were measured as a function of time. The sensor fabrication consisted of the following steps: a 1 mM solution of 11-mercaptoundecanoic acid (MUA) was made using ethanol 200 proof. Gold-plated quartz crystals were incubated in the MUA solution overnight. After incubation, the gold crystals were rinsed with ethanol to remove excess MUA [E. coli to the modified gold crystal was removed by incubating the sensor in 100 mM glycine solution. After each modification step, the crystal was inserted into the cassette for QCM measurements to determine the amount of individual mass added to the crystal.To design the mannose-derived QCM biosensor, the gold crystal was cleaned following a standard procedure, modified with the mannose derivatives or cess MUA . CarboxyE. coli were grown in TSB at 37 °C for 18 h. The bacteria cultures were centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the precipitates were dispersed in 0.1 M phosphate buffer (pH 7.02) [E. coli analyte. The bacteria concentration was adjusted with McFarland standards to achieve the desired concentration for optimal QCM detection. Subsequent serial dilution was performed to achieve the different concentrations of bacteria tested. Bacteria suspensions with a concentration of 103 to 106 CFU/mL were used as the samples. Tryptic soy broth (TSB) (30 g/L) was prepared by following the manufacturer’s instructions. pH 7.02) . This prE. coli on the QCM sensor surfaces was achieved via a micro-contact between the E. coli cells and the gold surfaces immobilized with mannose derivatives. The procedure entails pipetting an aliquot of 100 mL E. coli onto the modified gold crystal surface. The set-up was allowed to dry under nitrogen gas. The crystal was rinsed with ultrapure water to remove any unbound bacteria. The crystal was inserted into the cassette, and the change in frequency before and after the addition of the bacteria was measured (measured . The difn = 3). The mean and standard deviations were calculated for each triplicate of masses. The differences between the mean were subjected to a one-way analysis of variance and t-test, and p < 0.05 was significant.Experiments were performed in three replicates  following a revised protocol published elsewhere . The choThe synthesized ligands (4-NMBA and 4-TNM) were characterized by mass spectrometry (MS), as seen in In the case of 4-TNM, dimerization of the molecule was unavoidable due to the solvent effect, and utilizing organic solvents did not provide clear tandem, despite eliminating dimerization. The 4-TNM dimer gives 572 Da under positive ESI conditions. However, 577 Da was obtained due to the presence of amino groups. ESI-MS/MS degradation pattern revealed cleavage of water residues, coalescence of degradation products, and peaks from the dimer as expected. Nevertheless, the molecular weights of the mannose derivatives were predicted from ChemDraw (Perkins Software). The MS/MS peaks revealed possible fragmentation that agrees with ChemDraw prediction. From −1—(OH group), 1760 cm−1 (C=O), and 1716 cm−1 (-COOH) belong to D-(+) mannose [−1 and C–O at 1320–1210 cm−1 appear in both 4-NMBA and 4-TNM; however, the peak for the amide bond at 2100 cm−1 was present in only derivatized mannose but was absent in mannose. This observation confirmed the formation of both 4-NMBA and 4-TNM. Furthermore, the peak of the thiol group was at 2600 cm−1. As seen in FTIR analysis was conducted to determine the functional groups present in mannose and to compare them to mannose derivatives (4-NMBA and 4-TNM). In  mannose . An amidE. coli owing to the knowledge that aliphatic groups and aromatic rings attached to mannose enhance its selectivity and affinity to the FimH lectin.To determine the surface morphology of mannose derivatives for FimH lectin interaction, SEM characterizations were performed using JOEL 7001F SEM. The white crystal powder of the 4-NMBA and 4-TNM were deposited on a circular aluminum stub covered with double adhesive tape. The samples were then coated in a vacuum evaporator. The visualization was performed using an accelerating voltage of 5 kV . SEM imaThe NMR characterizations were performed to determine the amide bond formed after synthesizing the mannose derivatives. The 4-NMBA and 4-TNM showed a chemical shift of 6.7 and 7.9 attributed to the phenyl rings as compared to the shifts at 4.93 and 5.21 for only mannose. NMR data confirmed the successful synthesis of modified mannose, and the detailed characterizations are published in our work elsewhere .E. coli. The successful binding of E.coli to mannose derivatives on the surface of the quartz crystal has been demonstrated through changes in frequency and subsequent changes in mass determined by the Sauerbrey equation [E. coli. The sensor utilizes the 11-mecaptoundecanoic acid (MUA) linker due to its stability. Additionally, MUA is aliphatic and will interact with other aliphatic amino acids surrounding the carbohydrate-recognition domain of the FimH lectin. Furthermore, MUA was selected due to its simplified aliphatic structure with only one pKa value, and it provided good stability as a flexible linker [E. coli (CFU/mL). It is worth noting that a baseline was established to determine and understand subsequent frequency changes upon adding the modified ligands and the bacteria during the modification and detection steps, respectively. Each modification step added mass to the crystal, which led to an altered frequency response [E.coli via FimH lectin interaction. The determined mass from the resultant frequency changes of the mannose derivatives and FimH lectin interaction have been summarized in The two modified mannose, 4-NMBA, and 4-TNM were synthesized by reductive amination and were applied as a sensor platform due to the interaction between mannose derivatives and the FimH lectin of type 1 pili of equation . While oe linker . Aliphate linker . The senresponse . The charesponse . The finE. coli via carbohydrate and lectin interaction has been demonstrated with a mannose derivative immobilized QCM sensor. The fabrication of the QCM biosensor using 4-NMBA and 4-TNM was carefully achieved through a sequential modification. The purpose of each modification was to introduce aliphatic groups to the gold crystal surface before adding derivatized mannose. The aliphatic groups increase the selectivity of mannose derivatives to the FimH lectin. E.coli over the range of 1 × 103 CFU/mL to 1 × 106 CFU/mL. Each graph represents a different concentration of E. coli tested with 4-NMBA. The detection of E. coli. The frequencies from each modification were converted into masses using the Sauerbrey equation [E. coli cells attached to the crystal surface. E.coli over the concentration range of 1 × 103 CFU/mL to 1 × 106 CFU/mL. The detectable frequency for each modification step is represented by a letter at the end of each horizontal line. Specifically, (a) shows MUA addition to the ligands, (b) shows the EDC/NHS coupling, (c) shows the addition of the mannose derivative, (d) shows the addition of glycine, and (e) shows the addition of different concentration of equation ,21. FurtE. coli to the crystal resulted in a mass change of 3.796 ng for the 4-NMBA , Staphylococcus epidermidis, and Bacillus subtilis [E. coli, (gram(-)) due to enhanced derivatized mannose interaction with E. coli as compared to S. epidermidis (20%) and B. subtilis (15%). Fimbriae are short projections on the bacterial surface. FimH is an adhesive type 1 fimbria protein composed of two domains, namely an N-terminal (mannose-binding lectin domain) and a C-terminal pilin domain. FimH is expressed in enterobacteria (gram-negative E.coli) [Carbohydrate–lectin interactions at the surface of the host and pathogen reveal a crucial role in determining infections . The selsubtilis . The chosubtilis . It is e E.coli) ,39. The  E.coli) .n = 7). Briefly, the ligands (4-NMBA and 4-TNM) were successfully immobilized on the gold crystal along with the MUA linker molecule. The MUA was then activated by using EDC/NHS. The unbound ligand was removed by the addition of glycine. Finally, the prepared gold crystal was applied to detect E. coli. The results from the repeatability studies gave a standard deviation (SD) of 0.033 and 0.215 for 4-NMBA and 4-TNM, respectively. The reproducibility experiments (n = 7) were performed following a similar protocol and showed an SD of 0.023 (R2 = 0.9964) and 0.118 (R2 = 0.852) for 4-NMBA and 4-TNM, respectively. These results show the suitable application of the QCM biosensor for the detection of bacteria that express FimH lectins. To promote the practical application of mannose-derivatized QCM biosensors, reproducibility, and repeatability experiments were performed following the protocol described in the experimental section above. The repeatability experiment was performed using the same gold crystal after seven successive experiments  were synthesized via reductive amination and applied in the QCM biosensor fabrication. After the QCM experimentation, the 4-NMBA yielded a mass of 3.796 ng when it adhered to E. coli on a modified crystal. The 4-TNM ligand was synthesized from 4-aminophenyl disulfide and mannose, resulting in a mass of 314.2 ng when it interacted with E. coli. The sensitivity of the 4-NMBA ligand was determined to be 2.56 × 10−5 ng/mL, and the sensitivity of the 4-TNM ligand was determined to be 8.99 × 10−5 ng/mL. This study shows the suitability of the QCM biosensors to detect pathogenic bacteria as demonstrated in several reports.We have successfully designed and tested a new method for detecting


Fusobacterium nucleatum is associated with colorectal cancer (CRC), and identical F. nucleatum strains seemed to be detected in 75% of patients who exhibited the presence of F. nucleatum in both CRC and saliva samples in our previous study; however, the validation of strain identity and the development of a method for strain-level discrimination are required for etiological studies. We confirmed that F. nucleatum isolates obtained from CRC and saliva samples derived from patients with CRC originated from their identical strains using whole-genome sequencing. We evaluated the hypervariable region of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR–Cas) system in F. nucleatum strains isolated from the CRC and saliva specimens of CRC patients to develop a method for genotyping this bacterium at the strain level. The developed method consisted of two simple PCR steps to amplify the different lengths and sequences of the CRISPR–Cas regions from F. nucleatum strains using specific but common primers. This method could successfully detect identical strains present in both cryopreserved CRC samples and saliva obtained from the same CRC patient. Dynamic monitoring of F. nucleatum strains in saliva obtained from patients with colorectal adenoma before and after oral care showed interindividual variability in F. nucleatum strains during oral care. This study provided a simple and rapid method for comprehensively identifying F. nucleatum at the strain level in clinical samples, leading to a paradigm shift in CRC research, such as the investigation of pathogenic F. nucleatum strains and monitoring of pathogenic strains in clinical trials for preventing CRC recurrence. Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC. Fusobacterium nucleatum predominately colonizes the oral environment of humans and is pathogenically responsible for periodontitis   cases and 881,000 deaths are estimated to have occurred in 2018, accounting for about 1 in 10 cancer cases and deaths, and CRC ranks third in terms of incidence but second in terms of mortality  , and fibA (FadA) .F. nucleatum have been performed using metagenomics or 16S rRNA gene amplicon sequencing based on next-generation sequencing (NGS) technologies, but these NGS-based studies have limitations in terms of the resolution at the strain level. In our previous study, identical strains were detected in 75% of patients who showed the presence of F. nucleatum in both CRC and saliva specimens, suggesting that there is a high possibility that F. nucleatum in CRC originates in the oral cavity. In addition, only one to two F. nucleatum strains out of three to eight strains in saliva were present in CRC, suggesting the involvement of the strain-level pathogenicity of this bacterium in the development of CRC  and CRISPR-associated (Cas) (CRISPR–Cas) system comprises a novel mechanism of bacterial resistance to phage infection , which irculosis , 25, Camr jejuni , and Strpyogenes .F. nucleatum and demonstrated the effectiveness of this method in detecting strains of F. nucleatum in the CRC specimen and saliva obtained from CRC patients.Here, we investigated the use of CRISPR–Cas system-based PCR for bacterial strain typing of Randomly selected five pairs of isolates obtained from CRC and saliva of each CRC patient, which were suggested as identical strains by the arbitrarily primed polymerase chain reaction (AP-PCR) in our previous study , were anin silico analyses, the CRISPR–Cas system possessed by 26 strains of F. nucleatum in the NCBI database was classified into three subtypes, Types I-B, II-A, and III-A. We further categorized the Type I-B into two subtypes, Type I-B1 and Type I-B2, based on the similarity of Cas genes as well as the position of this system conserved on the genome  and Cas2 sequences of F. nucleatum clinical isolates  obtained from our previous study (F. nucleatum-strain typing PCR).Primer sets for CRISPR-associated regions are given in Table S3; these primer sets were designed based on the DNA sequences of 26 us study . In the F. nucleatum-strain genotyping PCR was performed to confirm the amplification of the CRISPR-associated regions of F. nucleatum-type strains. The result showed that these primer sets detected distinct bands for Type I-B1 and Type III-A, Type I-B2, Type I-B2, and Type II-A from F. nucleatum subsp. animalis JCM11025T, F. nucleatum subsp. nucleatum JCM8532T, F. nucleatum subsp. polymorphum JCM12990T, and F. nucleatum subsp. vincentii JCM11023T, respectively  obtained from five saliva specimens randomly selected from CRC patients  in our previous study . As a result, the same-size amplicon was detected from all isolates identified as identical strains by AP-PCR, excluding strain V3 that did not have all the CRISPR regions. These were confirmed as identical strains by this PCR analysis.Additionally, all isolates obtained from saliva of one CRC patient (Patient F) (19 isolates classified to four strains based on the AP-PCR patterns) were analyzed by F. nucleatum-strain genotyping PCR  and compared with PCR amplicon size estimated by genome sequences (Table S6). Subsequently, strain-specific amplicons with the same size as calculated from genome sequences were observed in each of the pairs, excluding pair of Isolates C-V3 and S-V3 that did not have all the CRISPR regions in genome sequence . Interestingly, we observed a difference of approximately 400 bp in PCR amplicons between Isolates C-P10 and S-P10, which originated from the identical strain using whole-genome sequencing, by both the F. nucleatum-strain genotyping PCR and the calculation using genome sequences. This suggests that this strain has relocated its habitat to oral or CRC, resulting in a different phage infection history.Furthermore, we analyzed paired isolates, which were confirmed to be originated from the identical strain using whole-genome sequencing, by F. nucleatum strains, according to our previous study , from which we successfully extracted genomic DNA in our previous study . From two patients (IDs O and Q), identical strains of F. nucleatum were detected in both CRC specimen and saliva by culture- and AP-PCR-dependent methods . In contrast, for Patient Q, identical strains were not detected in both CRC specimen and saliva by culture- and AP-PCR-dependent methods . The F. nucleatum-strain genotyping PCR analysis also did not detect any identical strains from both specimens obtained from this patient .For clinical application, to confirm whether this method can detect identical strains of  methods . The F. patients . In addich other , indicatF. nucleatum during clinical treatment, such as during the preliminary observations of the patients with colorectal adenoma. The dynamics of F. nucleatum strains in the saliva of patients with colorectal adenoma and periodontal disease, who were given oral care for approximately 3 months, were monitored via F. nucleatum-strain genotyping PCR. The results showed that one out of three strains detected in Subject 17 disappeared in the post sample, implying that this strain was markedly depleted in the abundance during oral care  and impairs the function of CD4+ T, CD8+ T, and NK cells; moreover, it reduces cytotoxicity, resulting in tumor escape from immunosurveillance , are involved in the development of CRC. In fact, there are differences in the presence of Fap2 and the adhesion rate to CRC cells at the strain level of F. nucleatum  using F. nucleatum-strain genotyping PCR. Subsequently, we detected identical F. nucleatum strains in both CRC and saliva samples in 66.7% (2/3) of the patients. However, this number of patients is too small, and further studies are needed.Isolating live bacteria from a biological sample using the culture method is essential for the study of bacteria at the strain level. However, culturing has a number of disadvantages: (i) time constraints due to the need to culture immediately after sampling and before death of bacteria and the time spent in cultivation and purification and (ii) bias because of skill proficiency, colony selection, and antibiotic sensitivity with selective agar and the presence of undetectable bacteria buried in colonies of dominant bacteria. These constraints and biases result in low reproducibility and may be difficult to apply to the medical setting. In contrast, a culture-independent metagenomics approach has promisingly improved enumeration of microbial species in clinical samples at the strain level . HoweverF. nucleatum and CRC, facilitating prevention of the recurrence of CRC and the suppression of deterioration of adenoma ; therefore, approximately 20% of strains would be undetected by this method. In addition, it is difficult to determine whether two strains are identical based on PCR amplicons of similar length in a sample. To confirm whether the strains are identical, DNA sequencing of the PCR products is required. In the samples analyzed in this study, we observed amplicons with undistinguishable size in Patient P. These amplicons corresponded to three isolates, i.e., C10b, C2, and S22. The AP-PCR pattern of C10b differed from that of C2 and S22. DNA sequencing of the amplicons showed that C10b amplicon had a sequence identical to that of C2 and S22 amplicons . This result suggests that F. nucleatum-strain genotyping PCR is a more sensitive and extensive method than AP-PCR for discriminating F. nucleatum at the strain level; however, further studies are warranted to confirm this finding. This method also has low quantifiability and needs to be combined with a quantitative analysis to address its physiological relevance.A limitation of this method is that F. nucleatum at the strain level. This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. Therefore, this method eliminates the disadvantages of culturing, such as time loss, skill bias, and low detection limits, and provides sensitive and stable data within a short time for high-throughput analysis. We believe that the new method would help in prevention of recurrence after CRC removal and suppression of carcinogenesis from adenoma.In this study, we demonstrated that some strains inhabit both the oral cavity and CRC tissues and developed a CRISPR–Cas region-targeted PCR method for the identification of F. nucleatum from the NCBI database (https://www.ncbi.nlm.nih.gov/) were downloaded (Table S2); CRISPR sequences were searched using CRISPRfinder program (https://crisprcas.i2bc.paris-saclay.fr/); and Cas genes were identified using the in silico Molecular Cloning Genomics Edition .The genome sequences of 26 strains of F. nucleatum subsp. animalis JCM11025T, F. nucleatum subsp. nucleatum JCM8532T, F. nucleatum subsp. polymorphum JCM12990T, and F. nucleatum subsp. vincentii JCM11023T, were purchased from the Japan Collection of Microorganisms of RIKEN BioResource Research Center. Clinical isolates previously identified as F. nucleatum obtained from the saliva of five randomly selected CRC patients  from our previous study  following the methods described in our previous paper , under anaerobic condition at 37°C for 48 h using AnaeroPack-Anaero .Type strains, us study  were useus paper . All FusF. nucleatum strains obtained from our previous study  was prepared using the SMRTbell template prep kit 2.0 . Sequencing data were produced with more than 100-fold coverage and assembled using the assembly program Canu v1.8  to obtaihttps://github.com/tseemann/snippy).SNVs between each pair of isolates and between non-paired isolates were analyzed using Snippy version (v4.6.0) . In addition, to confirm the presence of the CRISPR–Cas system, primer sets targeting the peripheral region of the CRISPR–Cas system  (TOYOBO), 300 nM of each primer set, and 5 ng or 1 µL of genomic/total DNA derived from F. nucleatum isolates or specimens, respectively. The thermal cycling condition was 94°C for 2 min; 25  or 30–40 (for specimen) cycles at 98°C for 10 s, each annealing temperature (Table S3) for 30 s, and 68°C for 3 min and 30 s. In second round of PCR, each of the four types of CRISPR regions was amplified by the 30-cycle PCR under same the condition using the primer sets for nested PCR with 1 µL of 100-fold  or 50-fold (specimens) dilution of the first PCR product as the template. The PCR products were electrophoresed on 0.7% (wt/vol) Tris-acetate-EDTA (TAE)-agarose gels and observed with a UV transilluminator Sayaca-Imager . For the analysis of multiple-strain mixture, equal amounts of each genomic DNA derived from multiple strains were used as a template.In the first round of F. nucleatum-strain genotyping PCR, the PCR amplicons were excised from agarose gel and purified using the FastGene Gel/PCR extraction kit . Both 5′- and 3′-ends of the purified amplicons were Sanger sequenced with the primers used for nested PCR amplification. Sequencing was performed by the FASMAC sequencing service . The sequences of the first 29 bases and the dozens of bases at the 3′ end were trimmed according to the sequencing chromatograms to around 600 nucleotides. The trimmed sequences were then analyzed by BLAST search against the nr/nt (for F. nucleatum subsp. nucleatum ATCC 25586) and the whole-genome shotgun contigs (for the other three type strains) databases with megablast algorithm. The repeat sequences in the obtained sequences were searched using the CRISPRCasFinder online program (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index) and visually confirmed.To verify the precision of AGTGAATTCGCGGTGAGATGCCA-3′) or D8635 (5′-GAGCGGCCAAAGGGAGCAGAC-3′)  .F. nucleatum-strain genotyping PCR amplicons were cloned into the pUC19 cloning vector using In-Fusion Snap Assembly cloning kits  according to the manufacturer’s instructions. A linearized vector with 16 bases homologous to the nested PCR product at both ends was created using the following primers: Type_I-B2_InFusion_F; 5′-GRCAAGTTAGAAAAAAGATCCTCTAGAGTCGACCTGCA-3′ and Type_I-B2_InFusion_R; 5′-TTTCATTTCTTGATTTGATCCCCGGGTACCGAGCTC-3′. Sequences homologous to the F. nucleatum-strain genotyping PCR products are underlined. The sequences of tissue F. nucleatum-strain genotyping PCR products and the PCR products from the corresponding isolates were sequenced by the primer walking method.g for 3 min), the supernatant was discarded, and the remaining 1 mL of viscous liquid at the bottom was collected as a saliva specimen and cryopreserved at −80°C. Intraoral care and medical guidance were provided by a dentist.Pre- and post-treatment saliva was collected from patients with both colorectal adenoma and periodontitis receiving oral care for 1–3 months. Five patients were randomly selected from participants. To collect saliva specimen, 10 mL of saline was gargled for 1 min and then collected. This was centrifuged (9,100 ×


Correction to: J Pharm Health Care Sci9, 2 (2023)10.1186/s40780-022-00272-9After publication of the original article  the authAccording to the statistical method used, this item should be included in multivariate analysis. However, the results for this clinical factor in the multivariate analysis were blank. The correct version of Table Accordingly, the corrected OR for younger age (< 55 years) related to pegfilgrastim-induced bone pain is 3.68 . Owing to the minimum alterations in OR and p-values in multivariate analysis, the authors believe that this correction does not significantly impact the study results.


High-purity eicosapentaenoic acid (EPA) ethyl ester (EPA-EE) can be produced from an integrated technique consisting of saponification, ethyl esterification, urea complexation, molecular distillation and column separation. In order to improve the purity and inhibit oxidation, tea polyphenol palmitate (TPP) was added before the procedure of ethyl esterification. Furthermore, through the optimization of process parameters, 2:1 , 6 h  and 4:1  were found to be the optimum conditions in the procedure of urea complexation. Distillate (fraction collection), 115 °C (distillation temperature) and one stage (the number of stages) were found to be the optimum conditions for the procedure of molecular distillation. With the addition of TPP and the above optimum conditions, high-purity (96.95%) EPA-EE was finally obtained after column separation. Long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are important components in healthy diets. So far, their health benefits have been widely reported, which include reducing triacylglycerols , anti-inn-3 LC-PUFAs are sold as soft gel capsules, aqueous emulsions and mixtures with vegetable oils, which are usually called omega-3 products. The commercial production of highly enriched n-3 LC-PUFAs is at present a major challenge for research. Single fractionation processes do not discriminate between different PUFAs. Therefore, several methods for preparing enriched fractions of n-3 LC-PUFAs have been developed, including controlled winterization , moleculSo far, commercial omega-3 products contain a high purity of total n-3 LC-PUFAs, but not EPA or DHA. As the most abundant omega-3 fatty acid in the central nervous system, DHA is important for the health of babies and pregnant women . MoreoveSchizochytrium cells in the upstream process, and then extract and purify the algal oils in the downstream process . m is the quantity (g) of fish oil and 100 is the conversion factor.According to the Chinese Standard GB 5009.227-2016 , the perg for 10 min. The obtained upper layer liquid was measured at 532 nm. The malondialdehyde concentration was converted to TBARS (ppm) by the following formula:532 is the absorbance value of the reaction mixture at 532 nm, and 2.77 is the conversion coefficient of malondialdehyde concentration into TBARS.According to the methods reported by John et al.  and Wang3OH was added. The mixture was refluxed for 5 min in an 80 °C water bath. The reaction was carried out by adding 2 mL of BF3-CH3OH . After 2 min, the reaction solution was extracted with n-hexane (1.5 mL). Before gas chromatography (GC) analysis, n-hexane containing fatty acid methyl ester (FAME) was filtrated through a 0.22 µm filter.According to the Chinese Standard GB 5009.168-2016 , the EPA0 (mg/g) in fish oil was calculated by the following formula:i is the influence factor of fatty acid methyl ester; Ai is the peak area of fatty acid methyl ester in the sample; AC11 is the peak area of triundecylin in the sample; ρc11 is the concentration of glycerol triundecylin solution (mg/mL); Vc11 is the volume of triundecylin added (mL); 1.0067 is the conversion coefficient of triundecylin to methyl triundecylin; m is the quantity of raw fish oil (mg); 10 is the coefficient of converting the content into the content in the sample per g; FFAMEi-FA is the conversion coefficient of fatty acid methyl ester to fatty acid.The specific parameters of GC analysis were as follows : the ini2 = 0.9992) and y = 507.95x + 6.65 (R2 = 0.9997), respectively.Standard solutions of EPA-EE and DHA-EE with different concentrations were prepared with n-hexane as the solvent, which were then filtered by 0.22 µm filter membranes. The detection method was the same as that described in 1 (mg/g) in the product was as follows:A certain quantity of ethyl fish oil product was mixed with 3 mL of n-hexane. The detection method was the same as that described in 1 is the quantity of ethyl fish oil products (g); C1 is the content of EPA-EE (or DHA-EE) in ethyl-esterified fish oil products (mg/g); M1 is the molar mass (mg/mmol) of EPA-EE (or DHA-EE); m0 is the quantity of raw fish oil (g); C0 is the content of EPA or DHA in raw fish oil (mg/g); M0 is the molar mass (mg/mmol) of EPA (or DHA).In the process of esterification, the esterification efficiency η (%) of EPA or DHA was calculated by the following formula:According to the procedure described by Zheng et al. , urea co2 and an evaporation surface of 5 dm2. The operation pressure was maintained at 0.3 mbar and the feed temperature was kept at 20 °C. The rotor speed was set at 200 rpm. The condenser temperature was set at 3 °C and the feeding flow was 1.0 ± 0.1 mL/min. The distillates and residues were obtained at different distillation temperatures  and different stages of distillation . Thus, based on the EPA-EE content and DHA-EE content of distillates and residues, the appropriate parameters, including distillation temperature and the number of stages in distillation, were obtained.Molecular distillation was performed in a KDL2 distiller . The instrument contains a condensing surface of 2 dmv/v, mL/mL) methanol–water as the mobile phase. The flow rate was 0.3 mL/min. The wavelength of detection was 220–270 nm. The injection volume was 140 µL.EPA-EE existed in the effluents from 49 min to 61 min. The purity of EPA-EE was measured through the GC method described in The column separation was used for the further purification of EPA-EE. The separation was performed on a C18 column  by isocratic elution with 92%  were considered significant.The results were calculated from parallel measurements and are given as means ± standard derivations. SPSS version 16.0 software  was applied for the statistical analysis. Differences between means were evaluated by one-way ANOVA (post hoc test: SNK) or independent samples t-tests. Comparisons that yielded Due to the high levels of PUFAs, including EPA and DHA, fish oils are more likely to be oxidized  in the pEuphausia superba) oil after the extraction process [As shown in  process , consistThe EPA-EE content, DHA-EE content and esterification efficiency were also calculated, and their values gradually decreased over storage time . ObviousIt is widely known that some vegetable oils such as sesame oil and hemp seed oil are rich in natural antioxidants. Many studies have shown that these natural antioxidants are effective in protecting oils from oxidation. For example, Shen et al. found that compared with oils extracted from red and white quinoa seeds, oil extracted from black quinoa seed contained higher PUFAs . PUFA coUrea complexation is a useful technique for removing saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) . BrieflyThe effect of the above three parameters on the EPA-EE content of ethyl-esterified fish oils is shown in Molecular distillation is one of the most effective purification methods to separate substances according to the difference in vapor pressure . In partIn order to further improve the content of EPA-EE, as well as reduce the content of DHA-EE in the products, the fish oils obtained from urea complexation were subjected to molecular distillation. As shown in Generally, the residues were collected in industrial production and some related research. For example, Fang et al. reported that after molecular distillation and urea complexation, the content of total PUFA increased from 307.2 mg/g (in crude fish oil) to 834.2 mg/g (in residue) . Wen et Furthermore, the results in this section clearly indicate that the EPA-EE and DHA-EE contents in distillates first increased, exhibited the maximum contents at the temperatures of 115 °C (EPA-EE) and 120 °C (DHA-EE), and then decreased with the increment in the distillation temperature. These results are consistent with other published findings. For example, Magallanes et al. reported that the content of EPA-EE in the distillate reached the maximum value (121.14 mg/g) at 120 °C, and then decreased  with the increase in distillation temperature . SimilarMolecular distillation is applied industrially to obtain fish oil and other products  . In manySchizochytrium limacinum oil after one-stage molecular distillation [In order to produce high-purity EPA, the distillate obtained from molecular distillation also requires subsequent chromatographic column separation. Obviously, such distillate should contain more EPA-EE and less DHA-EE. As shown in illation .Furthermore, it is essential to consider that two-stage (or more) molecular distillation performed with one or more types of distillation equipment are usually expensive . Given tThe combination of urea complexation  and moleIn the present study, based on the analysis of EPA ethyl ester (EPA-EE) contents, the optimum conditions of 2:1 , 6 h , 4:1 , distillate (fraction collection), 115 °C (distillation temperature) and one stage (the number of stages) to produce high-purity EPA-EE were obtained. Furthermore, the results of peroxide value, thiobarbituric acid reactive substances and esterification efficiency clearly indicate that adding tea polyphenol palmitate (TPP) before the procedure of ethyl esterification could effectively improve the purity and inhibit the oxidation. Thus, the technique with the addition of TPP to produce high-purity EPA-EE, which consisted of saponification, ethyl esterification, urea complexation, molecular distillation and column separation, was successfully applied.


The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99mTc-labeled-Duramycin in platelet imaging.Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with  The rol111In-Oxine (111In-8-hydroxyquinoline) and 99mTc-HMPAO (hexamethylpropylene amine oxime) became widespread in the 1980s [111In-Oxine still serves as the gold standard among the numerous compounds developed over the years [111In-ions redistribute to bind transferrin, and the imaging results would show bone marrow uptake, making platelet or free indium ion uptake indistinguishable. As recent tumor-related clinical research has shown, there are still unanswered questions regarding the in vivo biodistribution and trafficking of platelets. Therefore, the study of novel platelet-specific radiopharmaceuticals for easy access in pre-clinical models and not just for thrombosis diagnostics is a current topic.Besides routine platelet counts, the in vivo imaging of radiolabeled platelets also holds valuable diagnostic potential, which was recognized several decades ago. Thrombus formation, intravascular clotting, and different entities of peripheric circulation problems have been imaged in the clinic using radiolabeled platelets. Platelet accumulation in thrombi occurs not just during events associated with coagulation and endothelial damage but, evidently, in intra- and peri-tumoral blood clot sites as well. Hence, our intention to provide a clinically applicable new platelet radiolabeling methodology reflects the importance of new tumor imaging necessities and the need for in vivo platelet distribution imaging using isotopes in general. The first radiolabeling studies on platelets date back to the 1950s , and plahe 1980s ,8,9. Thehe 1980s . To overhe 1980s . Howeverhe years , but Ind99mTc using duramycin, which is a phosphatidylethanolamine-binding (PE) natural, polycyclic peptide antibiotic [99mTc, has previously been proposed as a novel radiopharmaceutical for apoptosis imaging [99mTc-HYNIC-Duramycin can be used for the stable radiolabeling of platelets without significant alteration in platelet activity and demonstrate the applicability of the thus-labeled platelets for in vivo small-animal SPECT/X-ray computed tomography (SPECT/CT) imaging investigations. As platelet membranes contain PE, the mechanism of radiolabeling is a simple membrane anchoring of the complexated 99mTc ion to the platelet membrane by duramycin binding to PE.In this paper, with this background, we report a novel method for a lipid-specific radiolabeling of platelets with tibiotic ,15,16. D imaging ,18,19. HThe reported use of a simplified platelet radiolabel should prove useful in studying tumor, CNS, and immunological research questions. We therefore aimed at performing and reporting this PE-duramycin-based isotope labeling of platelets in our experiments, presenting our results in the following original figures.Since the aim of our study was to test a novel radiolabeling procedure for platelets, we chose a separation protocol based on size-exclusion chromatography , which ensures the highest platelet purity ,21,22. I9 mL−1, which corresponds to approximately 20% of the initial platelet count in the concentrate. This yield can be explained mostly by loss due to non-specific adsorption during gel filtration, which was quantified in more detail during the radiochemical purification.Next, the size and concentration of purified platelets were determined with a novel microfluidic resistive pulse sensing instrument, which operates based on the Coulter principle . The obtBefore radiolabeling, the possible activation of platelets by duramycin was tested. The activation test was performed in a 96-well plate by measuring the absorbance as the function of time at 405 nm. 99mTc-HYNIC-Duramycin and incubating for 30 min at 30 °C with shaking. Purification was performed with the same gel filtration procedure that was used for the isolation of platelets. Radiolabeling of purified platelets was performed by simply mixing with 99mTc-HYNIC-Duramycin is not released as a single molecule from the surface of platelets but as attached to membrane fragments or to extracellular vesicles released from platelets during incubation.The plasma stability of the radiolabeling was assessed using 10-fold dilution of the labeled platelets in 0.8 μm filtered plasma, and the mixture was incubated at 37 °C with shaking for up to 3 h. At each time point, platelets were separated from the supernatant via filtration using either a 100 kDa MWCO filter or a 0.45 μm pore-size filter. Labeling stability percentages defined as the ratio of the activity of the platelet fraction and the filtrate are shown in 99mTc-HYNIC-Duramycin-labeled platelets in terms of labeled PE lipids per total number of PE lipids in one platelet can be estimated based on the platelet concentration, the activity concentration, the total phospholipid content, and the ratio of externally accessible PE lipids in platelets. The total phospholipid content of platelets was reported to be 3.5 mg/1010 platelets, and 25% of that consists of PE [−16 mol/platelet or 7 × 107 PE molecules/platelet. However, the externally accessible amount of PE in platelets is only 6.9% of the total PE lipids as measured using 2,4,6-trinitrobenzene sulfonate, which yields 4.8 × 106 labelable PE/platelet [99mTc-HYNIC-Duramycin molecules per platelet that can be calculated from the specific activity of the purified sample. In a typical experiment, we had 100 MBq to 300 MBq activity in 1 mL, with an estimated platelet concentration of 2 × 108 mL−1. Using the A = N·λ formula, where A is the activity of the sample in Bq, N is the number of decaying atoms, and λ is the decay constant defined as λ = ln 2/t1/2, in which t1/2 is the half-life of 99mTc (6 h), and the number concentration of 99mTc in the labeled platelet sample was approx. 3.1 × 1012 mL−1 to 9.3 × 1012 mL−1. These formulas yield 0.54–1.6 × 104 99mTc-HYNIC-Duramycin molecules per platelet, which means that approximately 0.3% to 1% of the PE molecules on the surface of the platelets are labeled.The specific activity of the ts of PE . Calculaplatelet . This ca99mTc-HYNIC-Duramycin has low tissue uptake and fast clearance from the circulation via the renal–urinary system [The in vivo imaging results of labeled platelets 3.5 h p.i. are shown in y system .111In-Tropolone and 111In-Oxine-labeled platelets in rabbits was reported by Dewanjee et al., who also observed significant liver uptake. Still, the activity in the blood was the highest after 24 h p.i. for both compounds according to this study [IIbβ3 (glycoproteins IIb/IIIa) receptor-specific peptide, should be mentioned [However, the use of radiolabeled platelets for the imaging of thrombosis was introduced in the 1970’s , and stuis study . Due to is study ,31 and bentioned . HoweverSingle-photon emission computed tomography (SPECT) radionuclide imaging can provide effective in clinical diagnostic applications because of its high sensitivity  and its ability to provide quantitative, whole-body imaging . This pl99mTc-based radiopharmaceutical than for 111In-oxime. The same dosimetry consideration applies to the half-life of 99mTc as compared to 111In. Therefore, this type of kit-based approach, coupled with also standardizable SEC separation as opposed to centrifugation-based hemolysis-prone cell/PLT whole-blood separations, can eventually be more readily trained and applied in clinical practice. The limitation of our duramycin-based platelet radiolabeling method might be represented in the current SEC purification protocol step. In a clinical application, a more seamless and less time-consuming, optimized version of this SEC purification method may need to be provided. To check the thrombosis-related clinical relevance, further studies are also warranted for the evaluation of experimental thrombus sites in pre-clinical models. However, this and other eventual pre-clinical tumor imaging results pertain to a different, more application-oriented in vivo model imaging study, as the results presented here point to a stand-alone new molecular method of obtaining and imaging stably radiolabeled platelets.This technetium-based platelet-selective labeling process might be realized in a more uniform and protocol-based way than In-oxime radiolabeling. However, the most important advantage of our method for further clinical use is that patient and staff dosimetry is significantly lower in any ® ACD-A, Greiner Bio-One Ltd., Budapest, Hungary), and a 10 μL prostaglandin E1 solution  was added. First, platelets were centrifuged at 800× g for 15 min at room temperature , and the supernatant was discarded. The pellet was gently resuspended in 1 mL HEPES-buffered Tyrode’s solution , and 0.5 mL of the washed platelet concentrate was further purified with size-exclusion chromatography (SEC) using a 3.5 mL gravity column filled with Sepharose CL-2B gel  equilibrated with THB solution. A total of 0.5 mL fractions were collected, and absorbance at 405 nm was measured with a BioTek Synergy 2 plate reader using 96-well plates [Washed human platelet concentrate was purchased from the Hungarian National Blood Transfusion Service  and was used within 24 h after arrival. A 9 mL platelet concentrate was mixed with a 1 mL ACD-A solution in a plastic tube (Greiner VACUETTEl plates . PurifieConcentration and size characterization of purified platelets was performed with microfluidic resistive pulse sensing (MRPS) using an nCS1 instrument  . SamplesSEC-purified platelets were diluted 10-fold in THB solution, and duramycin  was added to reach a final concentration of 0.35 μg/mL. Activation with thrombin at a 1 U/mL final concentration was used as a positive control. Mixing was performed in a 96-well plate and quickly placed in a BioTek Synergy 2 plate reader, and absorbance was measured at 405 nm at different time points for one hour [2, and 0.1 M sodium-cacodylate) at 4 °C overnight. For post-fixation, we applied 1% osmium tetroxide for 1 h. Next, the platelet pellets were dehydrated by graded ethanol series and then embedded in Spurr low-viscosity epoxy resin  according to the recommendation of the manufacturer. Ultrathin sections were made using a Reichert U3 ultramicrotome and collected on Formvar-coated copper grids. Uranyl acetate and Reynolds’ lead citrate were used to counterstain.Untreated platelets as a negative control, platelets treated with duramycin at 0.35 μg/mL concentration, and platelets activated with A23187 Ca-ionophore  as a positive control were investigated by transmission electron microscopy (TEM). Samples were centrifuged and fixed with a fixative solution  equipped with a Quemesa 11 MegaPixel bottom-mounted CCD camera .99mTc-pertechnetate ) solution was applied to the glass vial of the kit. The vial was incubated at 80 °C for 20 min to complete the labeling. Next, 0.25 mL of CL-2B-purified platelets in THB was mixed with 0.25 mL 99mTc-HYNIC-Duramycin (0.4 to 1 GBq) and incubated for 30 min at 30 °C with 200 RPM shaking in a neoMix cool thermomixer . Radiolabeled platelets were separated from free 99mTc-HYNIC-Duramycin by SEC using the same protocol as described in the previous section. The fractions corresponding to the void volume of the column (from 1 mL to 2 mL elution volumes) were pooled and used for testing the stability of the radiolabeling or for in vivo SPECT/CT investigations.HYNIC-Duramycin kit was purchased from Molecular Targeting Technologies . Radiolabeling was conducted following the manufacturer’s instructions. Briefly, 0.5 mL of freshly eluted 99mTc-HYNIC-Duramycin-labeled platelets with 2.25 mL of human plasma ). A total of 100 μL aliquots were placed in LoBind Eppendorf tubes and incubated at 37 °C with 200 RPM shaking in a neoMix cool thermomixer . At each time point, two separation procedures were performed in parallel with separate platelets from the free 99mTc-HYNIC-Duramycin: the samples were filtered with either 0.45 μm  or 100 kDa MWCO  centrifuge filters, and the activities of the flow through and retentates were measured in a dose calibrator .The radiolabeling stability test was performed by mixing 0.25 mL of Five healthy 6-week-old NMRI Nu/Nu mice (body weight between 22 g and 26 g) bred in the Animal House of Semmelweis University were used for biodistribution studies. Animals were allowed free access to food and water and were kept under humidity-, temperature-, and light-controlled conditions. All procedures were conducted in accordance with the ARRIVE guidelines and the guidelines set by the European Communities Council Directive (86/609 EEC) and approved by the Animal Care and Use Committee of the IEM and Semmelweis University (PE/EA/599-5/2021). A volume of 100 μL to 200 μL radiolabeled platelets in THB with an activity of 24.5 ± 1.6 MBq (Mean ± SD) was administered intravenously into the lateral tail vein. Mice were anesthetized with isoflurane for the whole duration of the imaging. SPECT/CT acquisitions were carried out using a nanoScan SPECT/CT  and started 3.5 h post-injection. SPECT and CT acquisitions were reconstructed using Nucline . Volumes of interest (VOIs) were manually delineated around selected organs . VOI uptake data are reported in % organ activity/injected dose and in standardized uptake value (SUV), which is defined as the ratio of the tissue radioactivity concentration and the injected activity divided by the body weight.99mTc-HYNIC-Duramycin is proposed. This method is aimed at possible overall use of platelet imaging with SPECT to further answer platelet distribution-related questions in vascular, tumor, immune, and aging biology. Our intention was not expressly the application of the radiolabeled platelets for any detection of thrombi but rather the report on the labeling and stability and the SPECT imaging possibility of the so-radiolabeled platelets for any biological purpose. Therefore, we need to note the limitations in our methods that need to be further tested in different biological pre-clinical whole-body imaging studies before the clinical translation of the method can be studied for patient diagnostics.In this paper, a novel method for the radiolabeling of platelets for in vivo SPECT/CT imaging using 99mTc-HYNIC-Duramycin.As the known mechanism of radiolabeling occurs, duramycin specifically binds to lipids with a phosphatidyl-ethanolamine head-group; hence, it provides a membrane-specific labeling. The performed activity tests indicated that in the concentration used for radiolabeling, duramycin does not cause platelet aggregation. The in vitro plasma stability of the labeling was found to be sufficient for in vivo studies. The performed small-animal in vivo SPECT/CT investigation revealed a good in vivo stability of the labeled platelets, which adumbrate the possible clinical applicability of the platelet-radiolabeling procedure with


Finally, the analysis of PCMC was carried out in multiple validated water samples using the synthesized SNG-C/CB-PANI sensor device, obtaining excellent results of recovery values (97–104%). The synergetic effect of polyaniline and carbon black leads to novel antifouling and electrocatalytic effects that improve the applicability of this sensor in sample analysis versus complex conventional devices.A new electrochemical sensor device has been developed through the modification of a polyaniline-silicon oxide network with carbon black (CB). Enhanced electrical conductivity and antifouling properties have been achieved due to the integration of this cheap nanomaterial into the bulk of the sensor. The structure of the developed material was characterized using Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and scanning electron microscopy techniques. Cyclic voltammetry was used to characterize electrochemically the Sonogel-Carbon/Carbon Black-PANI (SNG-C/CB-PANI) sensor device. In addition, differential pulse voltammetry was employed to evaluate the analytical response of the sensor towards sundry chlorophenols, common environmental hazards in aqueous ecosystems. The modified sensor material showed excellent antifouling properties, which led to a better electroanalytical performance than the one displayed with the bare sensor. Notably, a sensitivity of 5.48 × 10The online version contains supplementary material available at 10.1007/s00604-023-05740-z. The development of electrochemical sensor devices is currently devoted to simpler, cheaper, and more eco-friendly routes of synthesis of novel materials with enhanced analytical performance. Sonogel-Carbon electrodes (SNG-C), characterized by the application of high-energy ultrasound for sonocatalyzing the sol-gel process, stand out among handmade carbon ceramic sensors. Not only does this method of synthesis provide a highly competitive sensor through an eco-friendly and fast procedure, but a material susceptible to be modified with organic-inorganic receptors and biological recognition species to enhance its electroanalytical performance as well . WhereasNotably, conducting polymers and carbon nano allotropes are two promising types of compounds employed to improve conductivity, electrocatalytic effect, and/or antifouling properties in sensor devices –7. On thOn the other hand, carbon nanoallotropes have arisen in the last decades due to their properties and versatility, such as high-conductivity carbon black (CB). Remarkably, its employment as massive modifier of ceramic sensors has been exhaustively reported in recent years. CB stands out due to its electrocatalytic effect and low cost, among other interesting qualities , 13. UsuRecently, a new electrochemical sensor device, namely Sonogel-Carbon-Polyaniline (SNG-C-PANI), has been developed through the modification of the silicon oxide network of sonogel with PANI synthesized by sol-gel route assisted by high-energy ultrasound. The direct modification of the bulk material allows avoiding the typical problems of sensors modified with polymer coatings, such as the fast degradation of the layer and the adsorption of large molecules derived from the redox process. Moreover, this sensor has been successfully applied in the determination of chlorophenols; however, some disadvantages can be noticed, such as fouling phenomena after performing successive analysis and high capacitance values . ChloropIn this work, the massive modification of the original SNG-C-PANI bulk material with CB has been carried out, namely as Sonogel-Carbon/Carbon Black-Polyaniline (SNG-C/CB-PANI). CB has been selected as a modifier due to its low cost-effectiveness (c.a. 1 €/Kg), interesting electroanalytical properties, and absence of treatment requirement. The CB was successfully dispersed through the silicon oxide network. An improvement in the electrochemical performance of the sensor was expected with this modification thanks to its enhanced electrical conductivity and anti-fouling properties. The changes in the electroactive surface produced by fouling phenomena can affect electron-transfer kinetics and electrocatalysis, diminishing consequently the analytical sensitivity, stability, and operational lifetime . No repoThe structural and electrochemical characterization of the resulting SNG-C/CB-PANI material was carried out as well. The developed devices were tested with different chlorophenols, mainly 4-chloro-3-methylphenol or p-chloro-m-cresol (PCMC). The repeatability of the electrochemical measurements was studied with the modified sensor and compared with the unmodified one (with no CB). Finally, the developed sensor was successfully applied for the determination of PCMC in multiple untreated water samples, validating the results with HPLC as a reference technique. It must be highlighted the novelty of this work relies on that the proposed sensor device is simple, fast, eco-friendly, and cost-effective from the fabrication point of view. Moreover, other interesting features, such as versatility, stability, and easy mechanical renovation of the electrode surface also contribute to the performance of this new nanocomposite-based sensor. Finally, electrocatalytic and antifouling properties due to synergetic effect between PANI and CB in the composite could be considered as the main original contribution of this piece or research to improve electrical conductivity and electroanalytical performance, as well as to develop reliable and direct sample analysis. This kind of devices could be suitable to the analysis of several chlorophenols pollutants without significant interferences.d = 19–25 nm) from Cabot . All solutions were prepared with nanopure water obtained by passing twice-distilled water through a Milli-Q system . Glass capillary tubes, i.d. 1.15 ± 0.05 mm, were used as the bodies of the electrodes.All reagents employed were of analytical grade and used as received without further purification. Methyltrimethoxysilane (MTMOS), 4-chloro-3-methylphenol (PCMC), 4-chlorophenol, 2,4-dichlorophenol, and ascorbic acid were purchased from Merck . Ammonium persulfate (APS), potassium hexacyanoferrate (II), phenol, catechol, hydroquinone, resorcinol, and bisphenol A were from Sigma Aldrich . Aniline and 2,4,6-trichlorophenol were from Honeywell . Graphite powder was from Alfa Aesar  and carbon black powder (CB) (−2 cm2) as the working electrodes.The synthesis of PANI and sonogel materials were carried out using a high-energy ultrasound generator, Sonicator 4000 MISONIX  equipped with a 13-mm-diameter titanium tip, which provides 600 W as maximum output power. Electrochemical studies were made using an Autolab PGSTAT 20 potentiostat/galvanostat  connected with a personal computer and a 663 Metrohm VA Stand module. Processing data was made using GPES  software. The measurements were carried out in a three-electrode electrochemical cell at room temperature, with the following composition: Ag/AgCl/KCl 3 M as reference electrode, platinum wire as counter electrode, and the Sonogel-Carbon-PANI and Sonogel-Carbon/CarbonBlack-PANI electrodes (geometric area: 1.04 × 10Fourier-transform infrared spectra (FTIR) were recorded with a Fourier-transform infrared spectrophotometer IRAffinity-1S . X-ray diffractogram (XRD) were recorded with a diffractometer D8 Advance A25  using as source Cu Kα λ = 0.1542 nm and operating with 40 kV and 30 mA. Samples were prepared appropriately for DRX analysis. On the one hand, the sonosols of electrode materials prepared according to the procedure given in the “Scanning electron microscopy (SEM) was carried out on a Nova NANOSEM 50 , operating at 30 kV. For the SEM characterization, the filled tip of glass capillary tubes of SNG-C-PANI and SNG-C/CB-PANI electrodes were cut and inserted in the SEM sample holders with a hole previously drilled on its surface.The synthesis of SNG-C/CB-PANI was performed following a fabrication route previously reported with some modifications . FirstlyTo prepare the CB/graphite mixture, 1 g of a mixture of both materials was homogenized in a vortex mixer for 30 s at 3000 rpm. In this regard, several percentages of CB, ranging from 2.5 to 15% were assessed.The fabrication of the working electrodes was made following this methodology. Capillary tubes were filled with the mixture after the addition of graphite powder. Afterwards, carbon material was compacted and left to dry for 1 day. Before using, the electrode surface was polished gently using a Struers P1200 silicon carbide paper (Germany). Finally, a copper wire was inserted inside the capillary tubes to establish the electrical contact.−1 of scan rate and potential range from –0.2 to +0.6 V were set up. The electroactivity of the incorporated PANI was evaluated by means of CV in 1 M HCl at 50 mV·s−1 and a potential window ranging between –0.2 and +1.2 V.For the measuring procedure, a three-electrode electrochemical cell with 25 mL of solution was used. A previous characterization was carried out via cyclic voltammetry (CV) in presence of 5 mM potassium hexacyanoferrate (II) in 0.5 M potassium nitrate to select the best electrode material configuration in terms of electrochemical performance. As instrumental parameters, 50 mV·sDifferential pulse voltammetry (DPV) was chosen as the electroanalytical technique for the electrochemical determination of PCMC. The instrumental parameters were as follows: 8 mV as step potential, 50 mV of modulation amplitude, 0.4 s as interval time, and 0.05 s of modulation time. The current peaks obtained at 0.78 V (vs. Ag/AgCl/KCl 3 M) of working potential were evaluated considering a linear tangent baseline. To begin with, three buffer solution media at different pH were used to determine the optimal conditions for the detection of the target chlorophenol. The optimization study of optimal pH can be found in section S1 of Electronic Supplementary Material. Besides, in section S2 of supplementary information, capacities study can be found as well.Thereafter, calibration plots in presence of different PCMC concentrations ranging from 2 to 10 μM were recorded by adding adequate volumes from 1 mM PCMC stock solution, using DPV with the above-mentioned parameters. After adding the corresponding aliquot to the cell, the resulting solution was stirred for 30 s. Subsequently, the corresponding differential pulse voltammogram was recorded. The measurements for each concentration were done in triplicate to estimate the repeatability of the sensor. Likewise, this procedure was used in the calibration with other chlorophenols.Additionally, a study of the influence of different inorganic salts and electroactive species on the electrochemical signal of 7 μM PCMC oxidation was carried out. Particularly, 700 μM concentration of interferent was used to investigate the effect of sodium and calcium chloride, potassium and magnesium nitrate, and calcium and sodium carbonate on the analytical response of the device, whereas 70 μM concentration of cadmium and mercury chloride was also employed. A concentration of 7 μM was used for phenol, catechol, resorcinol, hydroquinone, ascorbic acid, and bisphenol A.Several water samples were collected from different sources: tap water, wells of Chiclana de la Frontera and El Puerto de Santa María, Igualeja’s spring, and Los Hurones reservoir . The analysis of PCMC-fortified water samples was carried out as follows: water sample was spiked with 25 μM of the analyte. Afterwards, the sample was diluted 10 times with ABS solution at pH 4 to reach 2.5 μM. Subsequently, electrochemical analysis with DPV was carried out employing a standard addition method by using concentrations ranging from 1 to 5 μM. Lastly, the electrochemical measurements were carried out in triplicate.Alternatively, the concentration of each sample was determined by HPLC and taken as reference values for comparison purposes. The used methodology can be found in section S3 from Electronic Supplementary Material.Firstly, as an initial attempt to modify the bare material (SNG-C-PANI), 600 mg of graphite and CB mixtures with different percentages were added to the sonosol. A pulverulent consistency could be appreciated in all modified materials, even in formulations with low proportions of CB. This effect may be attributed to the high specific surface area of the CB nanomaterial. Hence, aiming to accomplish the mechanical and compactness requirements for the final electrode material, several formulations of SNG-C/CB-PANI with 500 mg of graphite/CB mixture with different percentages of CB ranging from 1 and 50% were prepared. During the fabrication process, it was observed that SNG-C/CB-PANI formulations with a proportion of CB higher than 10% displayed worse consistencies; thus, proportions of CB ranging from 2.5 to 10% were electrochemically tested. The evaluation of their electrochemical performance, as well as of the SNG-C-PANI material (reference), was carried out using hexacyanoferrate (II) ion as the electrochemical probe.Two well-defined peaks corresponding to the oxidation and reduction processes of potassium hexacyanoferrate (II) were observed in the corresponding voltammograms Fig. A. ParticAdditionally, Fig. 3 symmetric deformation vibration at ≈ 1265 cm−1 (α), along with Si–O and Si–C stretching bands at ≈ 1110–1020 cm−1 (β) and ≈ 765 cm−1 (γ), respectively. These values are in consonance with the previous ones reported in the literature about Sonogel-Carbon materials [−1 and ≈ 1470 cm−1 that can be ascribed to C–N, C–C, and C=C stretching in the polymer [FTIR was used to examine the materials developed and corroborate the presence of PANI within the silicon oxide matrix. Hence, the infrared spectrum of powders from PANI 1), SNG-C (2), SNG-C/CB (3), SNG-C-PANI (4), and SNG-C/CB-PANI (5) materials was recorded  was used to study the electrode surface. For this purpose, micrographs of used and non-used SNG-C-PANI and Sonogel-C/CB-PANI electrodes were recorded and compared Figure SA and S3CMoreover, the surface of non-used electrodes Figures SE and S3GFirstly, SNG-C/CB-PANI and unmodified SNG-C-PANI materials were electrochemically characterized via CV. In both scenarios, the resulting cyclic voltammograms recorded in 1 M HCl solution Fig. A displayR2 = 0.994) suggests a diffusion-controlled process. Additionally, the value calculated for the slope of the plot of the logarithm of the anodic current peak versus the logarithm of the scan rate is 0.362 log A/log V·s−1, within 0.2 and 0.6 values, which corroborates a diffusion-controlled mechanism [Finally, the electrochemical evaluation of SNG-C/CB-PANI material using hexacyanoferrate process as electrochemical probe was carried out accordingly to the conditions mentioned in the Experimental section 2.4. The results of these studies are displayed in Fig. echanism .The analytical performance of the SNG-C/CB-PANI material was assessed via DPV by using PCMC as the benchmark analyte. The response of the bare material, SNG-C-PANI, towards this analyte has been also investigated for comparison purposes. Figure R2) of 0.996. Moreover, the sensitivity was calculated as the slope of the calibration curve. Therefore, for the SNG-C-PANI sensor the sensitivity was 3.05 × 103 ± 3.03 × 102 μA mM−1 cm−2, whereas for the modified SNG-C/CB-PANI sensor the sensitivity was 5.48 × 103 ± 3.05 × 102 μA mM−1 cm−2. The limit of detection (LOD) was also calculated as three times the standard deviation of the blank divided by the slope [Analytical calibration of both the original and the modified materials was carried out using PCMC as the benchmark analyte. DPV was used as the electrochemical technique, employing the instrumental parameters mentioned in the experimental section 2.4. The calibration plot was represented by the density current anodic peak versus different concentrations of PCMC. Accordingly, multiple additions were made to reach concentrations ranging from 2 to 10 μM, measuring each concentration in triplicate. The oxidation peak of PCMC, at a working potential of 0.78 V (vs. Ag/AgCl/KCl 3 M), increased with each addition, as can be observed in Fig. he slope . Hence, he slope . Thus, bIt should be stressed that the developed SNG-C/CB-PANI sensor exhibits better quality analytical parameters towards PCMC than the unmodified SNG-C-PANI sensor. Thus, the presence of CB in the bulk material has meaningfully improved the electrochemical performance of the sensor, which can be attributed to an increase of the charge transfer. Furthermore, the low variabilities of the current density values between measurements obtained with the modified sensor can be ascribed to the minimization of the surface fouling, which is in consonance with what was observed in the “Study of fouling phenomena effect” section. Hence, the presence of CB avoids the surface fouling of the modified material suffered by other sensor devices. The improvement that emerges from the modification of the material reduces the analysis time as well, due to the no necessity of electrochemical or mechanical renewal of the sensor surface after each measurement and boosts the easiness of the determination procedure.Besides, it is noteworthy to mention that the analytical parameters of the developed sensor are comparable to the results achieved with other sensors found in the literature. As shown in Table The SNG-C/CB-PANI sensor device has a substantial advantage versus other analogous sensors: no cleaning or later treatment is needed after analyte measurements due to antifouling properties. Sonogel–Carbon electrodes containing carbon black were developed by Pigani et al. and applied in the analysis of caffeic acid in instant coffee samples. However, an electrochemical cleaning process of 6 cycles (50 mV/s) with CV in KOH solution was necessary after each measurement . SimilarAdditionally, the calibration of other chlorophenols, 4-chlorophenol (4-CP), 2,4-dichlorophenol , and 2,4,6-trichlorophenol , was also carried out with the developed material. The analytical parameters of quality of the sensor are presented in Table SAlso, a brief comparison of the analytical parameters of the modified sensor toward these additional analytes and the performance of other sensors reported in the bibliography are shown in Table With the aim to assess the electroanalytical performance of the electrochemical sensor developed in this work, the influence of a wide range of ionic and metallic salts and other electroactive (mainly phenolic) species in the determination of PCMC was investigated. Accordingly, the following expression was used Eq. :PCMC and IPCMC+Int are the current peaks corresponding to PCMC oxidation in the absence and presence of the interferent evaluated, respectively. The results of this study are shown in Fig. where ILastly, analysis of real samples was carried out to ascertain the selectivity of the developed sensor and its applicability in complex matrices. Notably, the determination of PCMC in water samples is of great interest due to its role as an important environmental hazard in aquatic ecosystems. The US Environmental Protection Agency (USEPA) specify the water quality criterion for PCMC as a concentration level of 500 μg/L (3.5 μM) . TherefoThe electrochemical analysis of different water samples enriched with PCMC was carried out by the standard addition method, following the method reported in the Experimental section. Table SIn this work, a new sensor material based on the bulk modification of a ceramic-PANI composite with CB has been synthesized through a green and fast two-step ultrasound-assisted method. By reducing the amount of graphite powder from 600 to 475 mg, an optimized percentage of 5% of the nanomaterial CB has been also incorporated into the silicon oxide network with no volume contraction. The appropriate integration of CB in the bulk material has been proved through structural and electrochemical techniques, achieving the binding of both the nanomaterial and the polymer in the bulk structure.3 ± 3.05 × 102 μA mM−1 cm−2 greater than the one achieved with the unmodified material and competitive with those found in the literature. Furthermore, anti-fouling phenomena was exhibited by this material as a consequence of the inclusion of CB in its network, increasing its lifetime and versatility. Furthermore, a significant response of the sensor toward other chlorophenols  has been reported. Finally, it has been proved that this new device is useful in the determination of PCMC in natural untreated water samples, as referenced by HPLC studies, with recoveries values between 97 and 104%. As far as we know, the developed SNG-C/CB-PANI sensor is the first sensor device that is based on a material which combines the CB nanomaterial and the conducting polymer PANI. The synergetic effect of both materials leads to excellent antifouling and electrocatalytic properties that improve the electroanalytical parameters and allow to simplify sample analysis. This novelty heightens the new sensor device versus more complex and demanding  conventional sensors.The electrochemical sensor device developed showed great performance towards PCMC, displaying a sensitivity of 5.48 × 10ESM 1 (20.9 MB)